Antimicrobial resistance, virulence profile, and genetic analysis of ESBL-producing Escherichia coli isolated from Nile tilapia in fresh markets and supermarkets in Thailand

This study investigated the prevalence and antimicrobial resistance (AMR) of Escherichia coli (E. coli) in Nile tilapia from fresh markets and supermarkets. A total of samples (n = 828) were collected from Nile tilapia including fish flesh (n = 276), liver and kidney (n = 276), and intestine (n = 276). Overall prevalence of fecal coliforms (61.6%) and E. coli (53.0%) were observed. High prevalence of E. coli was found in the intestine (71.4%), followed by the liver and kidney (45.7%). The highest prevalence of resistance was commonly found against tetracycline (78.5%), ampicillin (72.8%), and sulfamethoxazole (45.6%) with resistance to only tetracycline (15.2%) as the most common antibiogram. The prevalence of multidrug resistance (MDR) (54.4%) and Extended-spectrum beta-lactamases (ESBLs) (5.7%) were examined. The predominant virulence genes (n = 158) were st (14.6%), followed by eaeA (0.6%). The blaTEM (73.4%), tetA (65.2%), and qnrS (57.6%). There is statistical significance between Nile tilapia from fresh markets and supermarkets. Based on logistic regression analysis, ampicillin-resistant E. coli was statistically associated with the phenotypic resistance to tetracycline and trimethoprim, and the presence of blaTEM and tetA (p < 0.05). Further investigation of AMR transference and their mechanisms is needed for AMR control.


Introduction
Antimicrobial resistance (AMR) is a crisis to global public health due to direct and indirect associated high morbidity and mortality of infected cases.In 2019, it was reported that there were 2.8 millions of AMR cases per year in the U.S. and AMR can cost 20 billion USD of healthcare-associated treatment [1,2].Without any effective control strategies, AMR will cause 10,000,000 deaths by 2050 with an estimated cost relating to AMR infection treatment around 100 trillion USD [3].The study in 2012 done in Thailand reported that AMR causes over 80,000 cases and 38,000 deaths per year [4].High prevalence of AMR in common human pathogens including Staphylococcus aureus, E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa has been reported with ranging from 27-52% in Thailand [5].It is estimated that the cost of treatment of these bacteria is 0.5 billion USD per year [4].
Both pathogenic and non-pathogenic bacteria can exhibit AMR.Bacteria that showed resistance equal to or more than 3 classes of antibiotic is called multidrug-resistant (MDR) bacteria, while bacteria that resist to all except 2 classes of antibiotics is termed as extensively drug-resistant (XDR) bacteria, and bacteria that resist to all antibiotics available is termed pandrug-resistant (PDR) bacteria [6].AMR bacteria usually harbor antibiotic resistant genes that coordinate with the group of antibiotics they resisted to.For example, bacteria that showed resistance to tetracycline normally harbor tetracycline resistance gene (tet), with 40 different genes have been currently characterized [7].E. coli is a bacterium in Enterobacteriaceae group, which is one of the most important bacteria that can cause diarrheagenic disease in humans through fecal-oral transmission.Therefore, it has been used as the determinant of fecal contamination in food production.Pathogenic E. coli that frequently contaminate food in Thailand are Enteropathogenic E. coli (EPEC), Enterotoxigenic E. coli (ETEC), Enteroaggregative E. coli, and Enterohemorrhagic E. coli (EHEC) or Shiga-toxin producing E. coli (STEC) [4,8].These pathogenic E. coli have specific virulence factors that are vital component to cause pathogenesis in humans and animals.The intimin producing gene, eaeA, corresponds to EPEC, while heat labile toxin gene (lt) and heat-stable toxin gene (st) correspond with ETEC, and shiga toxin 1 (stx1) and shiga toxin 2 (stx2) encoding genes correspond with STEC [9].E. coli can be an important reservoir of antibiotic resistance genes since they can exhibit more than one gene in the different plasmid types which can be transferred to neighbor bacteria through conjugation.Extended-spectrum β-lactamases (ESBLs) are a group of enzymes that bacteria produce and confer to resistance of most beta-lactam antibiotics, including monobactam, aztreonam, and cephalosporins.Moreover, ESBL-producing bacteria can exhibit co-resistance to multiple classes of antibiotics [10].The emergence of ESBL-producing Enterobacteriaceae, especially non-typhoidal Salmonella spp.and E. coli, became one of the crucial public health concerns, and subsequently fewer treatment options [11].ESBLs bacteria harbor specific resistance genes on plasmids [12].The most common genes found in ESBL bacteria in human and animal origin include bla TEM , bla CMY-2 , and bla OXA [13,14].Infection with ESBL bacteria can be more severe and prolonged hospital stay.In some severe ESBL-infected cases, last-resort antimicrobials are necessary for treatment of infected cases [15].
Nile tilapia (Oreochromis niloticus) is commonly cultivated worldwide due to its rapid growth, adaptability, and good commercial price [16].The production of Nile tilapia increases dramatically from 2,658 thousand tons in 2010 to 4,525 thousand tons in 2018 [17].Nile tilapia is a non-native fish species in Thailand and was introduced in 1965 and distribute to farmers by the Department of Fisheries [18].Therefore, most of the Nile tilapia sold in fresh markets and supermarkets in Thailand come from the fish farming in cages or ponds.In Thailand, Nile tilapia is one of the most important cultured fish in the country with a production of 337,500 metric tons [19].Exporting tilapia products from Thailand has been ranked among the top 6 countries in Asia, with 0.2 million tons in 2018 [17].To yield maximum tilapia production, an integrated fish farming system with high stock density of fish can lead to a wide range of bacterial diseases and subsequently contribute to heavy and prolonged use of antimicrobials in tilapia farms.This could contribute to promoting the selection of resistant bacteria and developing MDR bacteria [20].
Thailand's National Strategic Plan (NAP) on AMR 2017-2021 was established in 2016, and one of the main goals is to reduce the use of antimicrobials in animals, including the aquaculture sector.The Department of Fisheries of Thailand carried out the action plans by initiating AMR national surveillance program in fishery products, building microbiology laboratory capacity, and promoting antibiotic stewardship to farmers, in combination with farm biosecurity and disease prevention [21].Even though the distribution of AMR in aquaculture has been reported worldwide, available publications regarding AMR monitoring and surveillance in Nile tilapia remain largely unclear in Thailand.Therefore, this study aimed to explore the prevalence of fecal coliforms and E. coli in meat, kidney and liver, and intestine of Nile tilapia, to examine virulence genes and ESBL-producing E. coli, and phenotypic and genotype of AMR E. coli, and to identify the association between AMR, ESBLs, and virulence among E. coli isolates.
The scales of all Nile tilapia samples were aseptically removed at the dissected area and samples were divided into 25 g of fish meat (n = 276), one g of liver and kidney (n = 276), and one g of intestine (n = 276) to receive a total sample size of 828.All specimens were individually placed into a sterile plastic bag for further analyses.The animal study was reviewed and approved by the ethics committee of Chulalongkorn University Animal Care and Use Committee (IACUC; Approval No. 2031048).

Determination of fecal coliforms and E. coli
The samples were examined for fecal coliforms and E. coli followed by the United States Food and Drug Administration's Bacteriological Analytical Manual [22].Approximately, 225 ml of Buffered Peptone Water (BPW) (Difco, MD, USA) was added to fish meat samples, and 9 ml of BPW was separately added to liver and kidney, and intestine samples.The samples were homogenized using Stomacher 400 Circular lab blender (Seward, TX, UK).One ml of the mixed BPW suspension was added into 9 ml of lactose broth (Difco) containing a Durham tube.The samples were incubated at 37˚C for 24 h to observe gas production and turbidity.One loopful of positive lactose broth suspension was added to EC Broth (Difco) and incubated at 44.5˚C overnight.Turbid tubes with gas production were reported as fecal coliforms positive.A loopful of EC suspension was streaked on Levine-Eosin-Methylene Blue (L-EMB) agar (Difco) and MacConkey agar (Difco) plates.All the plates were incubated overnight at 37˚C.Suspected colonies of E. coli were flat and dark-centered colonies with or without metallic sheen on EMB agar and pink colonies on MacConkey agar.Suspected colonies on EMB and MacConkey agar were then biochemically confirmed using indole test and triple sugar iron agar (Difco) test.

ESBLs screening and confirmation
The detection of ESBL-producing E. coli isolates was performed using disk diffusion method [23].This method divides into two phases, screening and confirmation test.In screening test, E. coli isolates were cultured in Muller-Hinton agar (MHA) (Difco) at 37˚C overnight.Three antimicrobial disks of ceftazidime (30 μg), cefotaxime (30 μg), and cefpodoxime (10 μg) were placed on MHA agar plates and incubated at 37˚C overnight.Bacterial isolates showing the resistance to at least one antibiotic were further confirmed using combination disk diffusion method.

Detection of gene encoding virulence factors and antibiotic resistance
All E. coli isolates were determined as pathogenic E. coli including Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli (EPEC), and Shiga toxin producing E. coli (STEC), by specific virulence genes and tested for AMR genes by polymerase chain reaction (PCR).The DNA template was prepared by culturing the bacterial isolate in 5 ml Luria-Bertani (LB) broth (ThermoFisher Scientific, MA, USA) at 37˚C overnight while shaking at 120 rpm.The cultured bacteria were harvested and centrifuged at 3,000 rpm for 5 min.The pellet was resuspended with 200 μl sterile distilled water.The suspension was boiled for 10 min and then centrifuged at 5,000 rpm for 8 min.The supernatant containing DNA was collected and used as a template in PCR.PCR reactions containing DreamTaq PCR Master Mix (ThermoFisher Scientific) with a total volume of 25 μl containing 1 μM of the forward and reverse primer each were performed using T100™ Thermal Cycler (Biorad, CA, USA).Primers, annealing temperatures, and positive control isolates used for the detection of virulence genes and antibiotic resistance genes are summarized (Table 1).The PCR amplicon was mixed with StainIN™ GREEN Nucleic Acid Stain (highQu, Kraichtal, Germany), and then separated by 1.5% agarose gel electrophoresis in TAE buffer and visualized using Gel Doc™ EZ Systems (Biorad, CA, USA).The PCR product from positive isolates with no positive control was subjected to nucleotide sequencing and the sequences were analyzed using Nucleotide BLAST for gene confirmation.

Statistical analyses
Prevalence of AMR, MDR, virulence genes, and ESBL producing E. coli isolates were determined.Logistic regression analysis was used to determine the association among phenotype and genotype of AMR, virulence genes, and ESBL production.The dependent variable was ampicillin that is commonly used, while the independent variables included resistance phenotype and genotype, virulence genes, MDR, and ESBL production.A p-value and confidence intervals were adjusted for potential correlated data within type of market (fresh market and supermarket).Forward selection and backward elimination were used to choose candidates for multivariable analysis.Final regression models based on likelihood ratio test and p < 0.05 and two-sided hypothesis testing with 5% of significant level were used.Stata 14.0 (StataCorp, TX, USA) was used to perform all the statistical analysis.

Prevalence of fecal coliforms and E. coli in Nile tilapia
More than half of the Nile tilapia samples were positive to fecal coliforms and E. coli with overall prevalence of 61.6% (510/828) and 53.0%(439/828), respectively, where higher prevalence of both bacteria observed in samples from fresh markets than supermarkets.In fresh market, both fecal coliforms and E. coli were commonly found in the intestine (13.8% and 12.9%), followed by meat (12.7% and 10.1%), and liver and kidney samples (12.3% and 9.2%).However, fecal coliforms and E. coli isolated from the samples from supermarkets were mostly found in the samples from intestine (11.0% and 10.9%), the second most findings of both bacteria were in liver and kidney (6.6% and 6.0%), and least finding in fish meat (5.2% and 3.9%) (Table 2).

Genotypes of pathogenic and AMR E. coli
Genotypes of pathogenic E. coli using specific virulence genes revealed that ETEC was the most abundant pathogenic E. coli strains found in liver and kidney, and intestinal samples.EPEC was found in one intestinal sample, and none of pathogenic strains were found in any isolates from meat (Fig 1).

Multivariable logistic regression analysis
Phenotypic resistance to tetracycline and trimethoprim and the presence of bla TEM and tetA were significantly associated with the presence of ampicillin-resistant isolates (Table 4).A positive association was observed among E. coli isolates resistant to ampicillin and resistant to tetracycline and trimethoprim and the presence of bla TEM , while a negative association was examined in those isolates harboring tetA gene.This suggested that there is a mechanism linked to the resistance of ampicillin, tetracycline, and trimethoprim.However, inconsistency between phenotypic and genotypic resistance to tetracycline was determined among ampicillin-resistant isolates.

Discussion
Nile tilapia has been widely consumed worldwide, therefore intensive aquaculture farming system has been implemented to serve high demand both nationally and internationally.Even though Thailand is one of the major exporters of aquatic products [17], the epidemiological study of AMR and virulence genes in aquaculture is still limited.To strengthen AMR monitoring and surveillance in aquaculture, characterization of the phenotypic and genotypic resistance, ESBL production, and virulence genes of E. coli isolated from Nile tilapia was performed.E. coli has been commonly used as priority organism to monitor AMR in humans and animals.The contamination of resistant E. coli in Nile tilapia can occur from fish production site through to consumption.This study emphasized the high prevalence of fecal coliforms and E. coli contamination in Nile tilapia sold in fresh markets and supermarkets.Various sources of potential contamination of AMR E. coli should be carefully monitored throughout the production pipeline, as it could occur at any stage.The contaminant from the farm may arise from fecal material in water or soil used in the raising pond.During transportation and in fresh market, the contamination may originate from dust, ice or storage water.In the selling area, contaminants could even be found on knives or the display table.The result also indicated a high prevalence of AMR E. coli in intestine of the fish, therefore the contamination of AMR E. coli in fish flesh might come from the intestine during the preparation process.Further investigation is needed to assess the hygienic practices of the handler and the bacterial contamination on tools and surfaces used in the selling area.This is crucial for determining the sources of contamination and associated risk factors, leading to a better understanding, and improved planning for prevention and control measures.The overall prevalence of fecal coliforms and E. coli were higher in fresh markets (fecal coliforms 38.8%; E. coli 22.8%) compared with supermarkets (fecal coliforms 32.3%; E. coli 20.8%).These findings agreed with previous studies that high prevalence of E. coli in Nile tilapia was observed [35,36].In addition, the result was similar to previous study in Thailand, which reported high prevalence of E. coli and K. pneumoniae collected from fish markets compared to fish farms [37].Higher prevalence of E. coli in fresh markets can reflect inappropriate storage, cross-contamination, or poor hygienic condition.At farm level, contaminated water or cross-contamination during harvest and transportation can contribute to inconsistent levels of E. coli [38,39].
The highest prevalence of E. coli was observed in intestinal samples in Nile tilapia from both fresh markets and supermarkets in this study.The findings agreed with the study in Egypt [40].This study reported EPEC was found in intestine sample only (Fig 1 ), which was in accordance with the studies reported high prevalence of EPEC in fish sold in fresh markets in Brazil [41,42].Very few studies have observed the presence of ETEC in Nile tilapia, but this study reported a high prevalence of ETEC in intestine, and liver and kidney samples (Fig 1).This is concerning since the consumption of tilapia contaminated with ETEC can lead to diarrhea, even though the symptoms are not life threatening.Interestingly, the high prevalence of E. coli found in fish meat was determined in this study, however, none of them were pathogenic E. coli.Regardless that fish meat has been noted as a sterile sample, the presence of E. coli in fish meat in this study may be because microorganisms can penetrate from degradation of digestive system to muscle fiber when fish are restrained and caught during harvest (40).From food safety perspective, the national and international limits of E. coli in fish for human consumption should not exceed 10 MPN/g [43,44].However, this study failed to quantify the level of E. coli in meat samples.Therefore, the enumeration of fecal coliforms and E. coli should be further investigated for public health purposes.The contamination of AMR bacteria in Nile tilapia in retails is of public health significance.Nile tilapia is typically cultured in an earth pond, relying on water from local canals for pond filling, draining, and maintaining water level of the pond through water inlet and outlet.However, the low concentrations of antimicrobials in the pond and canal water indicated that the effectiveness of Thailand policies of antimicrobial use in aquaculture.Rather than focusing solely on the level of antimicrobial usage, attention has been directed towards the correlation between fecal bacteria and genes resistant to antibiotics, revealing important insights [45].Therefore, the contamination of the AMR E. coli in Nile tilapia was likely to originate from human E. coli and pollution from inadequate water treatment which contaminated in the canals water [46].Occurrence of AMR in tilapia had been reported worldwide [35].Antimicrobial use in humans, and animals, including aquaculture can drive the bacteria resistance.Improper use of antimicrobials as growth promotors and prophylaxis in aquaculture has been documented, and evidence suggested that it might lead to AMR accumulation in the fish and surrounding environment [47,48].Despite limited evidence of antimicrobial use in Nile tilapia [49], this study reported the prevalence of resistant E. coli isolates at 36.0% and high prevalence of MDR E. coli at 54.4%.Many studies have shown that most of the bacteria, especially E. coli and Salmonella spp.isolated from tilapia are MDR [48,50,51].Generally, antimicrobial use in aquaculture is varied in each region depending on local regulations.This study reported high resistance to tetracycline, beta-lactam, and sulfamethoxazole, which correlated with antibiotic groups highly use in aquaculture in Thailand [52].The resistance pattern was similar to the study of AMR E. coli isolated from tilapia collected directly from aquaculture farms in Malaysia [51].This might indicate the fish cultivation sites as one of the major sources of resistant organisms.However, it is worth noting that certain resistance genes can be acquired naturally by bacteria regardless of the use of antibiotics [53].Therefore, the result calls for consistently monitoring of AMR E. coli and other pathogenic bacteria in aquaculture.The low prevalence of ESBLs-producing E. coli was less than 6.0%, which was in agreement with the low prevalence of ESBLs-producing E. coli reported in tilapia in Vietnam when compared with higher prevalence of ESBL E. coli in other fish species [50].Most of the ESBLs-producing E. coli isolates were MDR, but none of the isolate were pathogenic E. coli.ESBLs-producing E. coli isolates that harbored resistance and MDR genes are of public health threat, since these isolates can facilitate resistance to other bacterial species impacting dissemination and evaluation of AMR in the environment [54].Even though the prevalence is low, surveillance of the ESBLs-producing bacteria should be conducted regularly and consistently to monitor the culture standard and prevent the spread of the bacteria.
Based on logistic regression analysis, the ampicillin-resistant isolates were more likely to resist tetracycline 25.3-fold (OR = 25.3, e 3.232 , p = 0.006) and trimethoprim 30.2-fold (OR = 30.2,e 3.408 , p = 0.023) than those susceptible isolates.In addition, the E. coli isolates showing ampicillin resistance were more likely to contain bla TEM gene (OR = 266.3,e 5.584 , p < 0.0001).This finding was supported by previous study indicating an interaction between β-lactamase genes (bla TEM-1 ) and tetracycline efflux pump, which generally encoded on transmissible elements, including plasmid, integrons, and transposons [55].It is evidence that coselection of ampicillin and trimethoprim can occur through mobile genetic elements [56].However, this study observed the negative association between ampicillin resistance and the presence of tetA genes (OR = 0.137, e -1.986 , p < 0.0001), meaning that the ampicillin-resistant isolates were less likely to confer tetA 7.30 times (1/0.137= 7.30) than those susceptible ones.This result addressed that even though the AMR is usually involved in genetic changes, the result showed that genotype of AMR E. coli isolates did not correspond well with their resistant phenotypes.Evidence from past studies reported similar results in E. coli and Salmonella spp.[33,57].This might be the result of limitation of the detection method by PCR.However, the resistance of the bacteria without corresponding genotype is existed and labelled as solely phenotypic resistance, which is usually found during infection process due to the stationary growth phase or persistence [58].However, the phenomenon is not considered in the antimicrobial susceptibility test.This suggested that there might be other genes or regulatory factors that influence resistant phenotype of the bacteria.To overcome the limitation and explore other genes in the bacterial genome, whole genome sequencing using next-generation sequencing might be able to give all information on the resistance genes in the bacteria.Resistant pathogenic E. coli in Nile tilapia can contribute to a difficult to treated gastrointestinal infection in consumers.Other resistant E. coli, on the other hand, can become a reservoir of the resistant genes and transfer the genes to other pathogenic bacteria via horizontal gene transfer [59,60].The presence of AMR E. coli in Nile tilapia in the retail is concerning since they can serve as reservoir of resistant genes and disseminate them to other bacteria.Hence, AMR surveillance and monitoring system in the environment, observation of AMR in aquatic animals sold in markets is needed.

Conclusion
Taken together, this study addressed the circumstance of circulated resistant E. coli in Nile tilapia sold in fresh markets and supermarkets in Thailand.This study identified that fish intestine was a dominant source of fecal coliforms and E. coli contamination and could be used as a target sample for AMR study in aquaculture.Pathogenic and resistant E. coli contaminated in Nile tilapia can pose a serious public health threat.This result can be used to support further investigation to identify the potential source of resistant bacterial contamination.Study of AMR in aquaculture under One Health is also needed to generate baseline epidemiological data for effectively prevent and control AMR.Furthermore, implementation of better sanitation and control spread of AMR bacteria in the environment are recommended.Continuing monitoring and surveillance of AMR will assist to strengthen AMR national strategic plan in aquaculture.

Table 2 . Prevalence of fecal coliforms and E. coli from Nile tilapia.
a calculated from grand total E. coli isolates b calculated from grand total resistant E. coli isolates https://doi.org/10.1371/journal.pone.0296857.t002